The potential of antifolate drugs in the treatment of malignant hepatoma will be investigated. The approach to be used will be a search for exploitable biochemical differences between experimental hepatomas of the rat and normal rat liver and other normal rat tissues such as bone marrow and intestinal epithelium. The biochemical factors to be studied will include enzymes of folate metabolism, reduced folate cofactor pools, ribonucleotides and deoxyribonucleotides, and cellular contents of free amino acids related to one-carbon group transfer reactions. Where biochemical differences are found between the malignant and normal tissues, attempts will be made to devise selective chemotherapy based upon these differences. These experimental chemotherapy studies will be conducted initially in cell culture, using five established rat hepatoma lines. The most effective drug combinations will be used for further study in vivo. These animal experiments will include measurements of cellular levels of folate cofactors, and nucleotides and nucleosides in drug-treated tissues (normal and malignant) and survival prolongation and tumor mass measurements. Emphasis will be placed on combinations of antifolates with antipyrimidines. Selective use of antimetabolites requires a fuller understanding of metabolic regulation, and a number of outstanding problems in regulation of mammalian folate metabolism will be considered, specifically the regulation of deoxyuridylate production and utilization, and the interactions between nucleotide salvage pathways and folate reactions. The possibility that methotrexate and other antifolates may cause single-strand breaks in DNA will be explored. Based upon biochemical and pharmacological properties of the individual drugs under study, the technique of metabolic simulation with mathematical models will be used to predict drug antagonism or synergism; the predictions will be tested in the tissue culture and animal hepatomas.